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Abstract:

Method that includes providing plurality of test sites each having first
and second layers respectively including inorganic first and second
surface sites forming parts of interior of a well, the surface sites
having positions and thicknesses being configured for locating thereon
portion of unidentified amino acid-containing molecules; exposing each of
a first plurality of the test sites to a fluid containing a different one
of plurality of pre-identified amino acid-containing molecules and
determining bonding signatures onto each of first plurality of test
sites; exposing each of second plurality of test sites to another fluid
containing unidentified amino acid-containing molecule and determining
bonding signatures onto second plurality of test sites; and comparing
bonding signatures to determine or exclude identity of unidentified amino
acid-containing molecule.

Claims:

1-16. (canceled)

17. A method, comprising: providing a plurality of test sites, each of
the test sites having a first layer including an inorganic first surface
site and a second layer including an inorganic second surface site, each
of the first and second surface sites forming parts of an interior of a
well, the first surface site having a position within the well and having
a thickness, the position and the thickness being configured for locating
thereon a portion of an unidentified amino acid-containing molecule, the
second surface site having another position within the well and having
another thickness, the another position and the another thickness being
configured for locating thereon another portion of the same unidentified
amino acid-containing molecule; exposing each of a first plurality of the
test sites to a fluid containing a different one of a plurality of
pre-identified amino acid-containing molecules and determining a bonding
signature of each of the different one of the plurality of pre-identified
amino acid-containing molecules onto each of the first plurality of the
test sites; exposing each of a second plurality of the test sites to
another fluid containing the unidentified amino acid-containing molecule
and determining a bonding signature of the unidentified amino
acid-containing molecule onto each of the second plurality of the test
sites; and comparing the bonding signatures of the plurality of the
pre-identified amino acid-containing molecules with the bonding signature
of the unidentified amino acid-containing molecule, to determine or
exclude an identity of the unidentified amino acid-containing molecule.

18. The method of claim 17, wherein the providing a plurality of test
sites includes providing each of the test sites as having a third layer
being located between the first and second layers, the third layer
including an inorganic third surface site, each of the third surface
sites forming a part of the interior of the well, the third surface site
having a further position within the well and having a further thickness,
the further position and the further thickness being configured for
locating thereon, a further portion of the unidentified amino
acid-containing molecule.

19. The method of claim 17, wherein the providing a plurality of test
sites includes providing each of the test sites as having a third layer
between the first and second layers, the third layer including a third
surface site, each of the third surface sites forming a part of the
interior of the well, the third surface site having a further position
within the well, the further position being recessed in the interior of
the well away from the position of the first surface site and away from
the another position of the second surface site.

20. The method of claim 17, wherein the providing the second plurality of
the test sites includes removing the plurality of the pre-identified
amino-acid containing molecules from the first plurality of the test
sites, and using the first plurality of the test sites as being the
second plurality of the test sites after the removing.

21. The method of claim 17, wherein the providing a plurality of test
sites includes providing some of the inorganic first and second surface
sites as being semiconductor surface sites.

22. The method of claim 17, wherein the providing a plurality of test
sites includes providing some of the inorganic first and second surface
sites as being group III-V semiconductor surface sites.

24. The method of claim 17, wherein the providing a plurality of test
sites includes providing some of the inorganic first and second surface
sites as having a metallic insulator composition including an oxide, a
carbide, a boride, a nitride, or a sulfide.

25. The method of claim 17, wherein the providing a plurality of test
sites includes providing some of the inorganic first and second surface
sites as including silicon nitride, silicon dioxide, aluminum oxide, zinc
oxide, beryllium oxide, ferrite, zirconium oxide, boron carbide, silicon
carbide, or magnesium diboride.

27. The method of claim 17, wherein the exposing each of the second
plurality of the test sites to another fluid containing the unidentified
amino acid-containing molecule includes depositing into the well, a fluid
containing the unidentified amino acid-containing molecule as being an
amino acid, a polypeptide, or a protein.

28. The method of claim 17, wherein the providing a plurality of test
sites includes providing one of the plurality of the test sites as
including the well and providing another one of the plurality of the test
sites as including another well.

29. The method of claim 17, wherein the determining the bonding
signatures of each of the amino acid-containing molecules includes
detecting optical absorption and reflectance at each of the plurality of
the test sites.

30. The method of claim 17, wherein the providing a plurality of test
sites includes providing the inorganic first and second surface sites as
having different inorganic compositions.

31. The method of claim 17, wherein the providing a plurality of test
sites includes providing each of the test sites as including a fourth,
layer having the well therein, the first and second layers extending
laterally across a part of a bottom of the interior of the well, the
inorganic first and second surface sites being exposed to the interior of
the well, each of the test sites further including a first conductor and
a second conductor, the first and second conductors being located to
apply a voltage across a portion of the well, wherein the first and
second layers are nonadjacent and the first and second surface sites are
distant from the fourth layer.

32. The method of claim 31, wherein the providing a plurality of test
sites includes providing the inorganic first and second surface sites as
having different inorganic compositions.

33. The method of claim 31, wherein the providing a plurality of test
sites includes providing the first and second conductors as extending
along a surface of the fourth layer such that each of the conductors has
an edge adjacent the well, the conductors being located to apply a
voltage across the well; and wherein the determining a bonding signature
of each of the amino acid-containing molecules includes placing the first
and second conductors in electrical communication with an external source
of a voltage and detecting a conductivity across the well between the
first and second conductors.

34. The method of claim 31, wherein the providing a plurality of test
sites includes providing the first and second conductors as respectively
being connected to non-exposed parts of the first and second layers, and
wherein the determining a bonding signature of each of the amino
acid-containing molecules includes placing the first conductor in
electrical communication with an external source of a voltage and placing
the second conductor in electrical communication with another external
source of another voltage.

35. The method of claim 31, wherein the providing a plurality of test
sites includes providing each of the test sites as having a third layer
being located between and separated by portions of the fourth layer from
the first and second layers, the third layer extending laterally across
and forming a part of the bottom of the interior of the well, the third
layer including an inorganic third surface site being distant from the
fourth layer and exposed to the interior of the well, the third surface
site having a further position within the well and having a further
thickness, the further position and the further thickness being
configured for locating thereon a further portion of the unidentified
amino acid-containing molecule.

Description:

FIELD OF THE INVENTION

[0001] This invention relates to the field of apparatus for the detection,
identification, characterization and further analysis of biological
materials.

BACKGROUND OF THE INVENTION

[0002] Tremendous progress has been made over several decades in the study
of biological materials ranging from amino acids, to proteins, to the
entire human genome. In spite of the great strides that have already been
made, cost-effective and timely analysis of biological materials
frequently is still not a reality. For the myocardial infarction victim
waiting in the hospital emergency room or undergoing a heart bypass
operation, the time needed for conventional blood analysis in order to
detect the telltale enzyme signature of a heart attack may be too long.
Myriad other circumstances can be observed in which analytical test
results on biological materials simply take too long to generate, aren't
available where needed, and cost too much. Furthermore, conventional
diagnostic tests typically are encumbered by their own particular
collection of analytical inadequacies, leading to false positive and
negative results at levels that are both intractable and statistically
significant.

[0003] Accordingly, there is a continuing need for analytical apparatus
that can be used to detect, identify, characterize and otherwise analyze
biological materials, including for example amino acids and proteins.

[0005] In one embodiment, an apparatus is provided, comprising: a first
surface site comprising a first substantially inorganic surface having a
first chemical composition selected from the group consisting of metals,
semiconductors, insulators, and mixtures thereof, said first surface
positioned within a polypeptide bonding region and having a selective
bonding affinity for a polypeptide; a plurality of first interlayers
between which said first surface site is interposed; a first distal site
end on said first surface site and distanced from said first interlayers,
said first surface being provided on said first distal site end; said
first surface site and said first interlayers being interposed between
first and second supports; first and second conductors provided on said
first and second supports and having respective first and second distal
conductor ends positioned within said polypeptide bonding region; said
conductors being capable of applying an external voltage potential across
said polypeptide bonding region.

[0006] In another embodiment, an apparatus is provided, comprising: a
first surface site comprising a first substantially inorganic surface
having a first chemical composition selected from the group consisting of
metals, semiconductors, insulators, and mixtures thereof, said first
surface positioned within a polypeptide bonding region and having a
selective bonding affinity for a polypeptide; a plurality of first
interlayers between which said first surface site is interposed; a first
distal site end on said first surface site and distanced from said first
interlayers, said first surface being provided on said first distal site
end; and a first conductor in electrical communication with said first
surface site, said first conductor positioned for electrical
communication with a source of an external bias voltage.

[0007] In a further embodiment, a method of making an apparatus is
provided, comprising the steps of: providing a first surface site
comprising a first substantially inorganic surface having a first
chemical composition selected from the group consisting of metals,
semiconductors, insulators, and mixtures thereof, having a selective
bonding affinity for a polypeptide; positioning said first surface within
a polypeptide bonding region; interposing said first surface site between
a plurality of first interlayers; providing a first distal site end on
said first surface site and distancing said first distal site end from
said first interlayers; providing said first surface on said first distal
site end; interposing said first surface site and said first interlayers
between first and second supports; and providing first and second
conductors on said first and second supports, having respective first and
second distal conductor ends positioned within said polypeptide bonding
region.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 shows a top view of an embodiment of an amino acid detection
and identification apparatus;

[0009] FIG. 2 shows an array of exemplary control test data for
polypeptides, generated using the amino acid detection and identification
apparatus of FIG. 1;

[0010] FIG. 3 shows another array of exemplary control test data for
polypeptides, generated using the amino acid detection and identification
apparatus of FIG. 1;

[0011]FIG. 4 shows a further array of exemplary control test data for
polypeptides, generated using the amino acid detection and identification
apparatus of FIG. 1;

[0021] FIG. 14 shows a side view of an embodiment of an apparatus
embodying modifications of the apparatus shown in FIG. 12;

[0022] FIG. 15 shows an application of the apparatus shown in FIG. 12 for
the detection and identification of a target polypeptide macromolecule in
which an antibody for the macromolecule is employed;

[0023] FIG. 16 shows a perspective view of an embodiment of an additional
amino acid detection and identification apparatus;

[0024] FIG. 17 shows steps of a method for making the amino acid detection
and identification apparatus of FIG. 1;

[0025] FIG. 18 shows steps of a method for using the apparatus of FIG. 1
for detection and identification of an unknown polypeptide in a fluid;

[0026] FIG. 19 shows steps of a method for making the amino acid detection
and identification apparatus shown in FIGS. 8 and 9;

[0027] FIG. 20 shows steps of a method for using the apparatus of FIGS. 8
and 9 for detection and identification of an unknown polypeptide in a
fluid;

[0028] FIG. 21 shows steps of a method for making the amino acid detection
and identification apparatus shown in FIGS. 12, 13 and 14;

[0029] FIG. 22 shows steps of a method for using the apparatus of FIGS.
12, 13 and 14 for detection and identification of an unknown polypeptide
macromolecule in a fluid; and

[0030] FIG. 23 shows steps of a method for making the amino acid detection
and identification apparatus shown in FIG. 16.

DETAILED DESCRIPTION

[0031] Apparatus are provided for the detection, identification,
characterization, and other analysis of biological materials. The
biological materials to be analyzed can include, for example, amino
acids, polypeptides, and proteins. The detection apparatus comprise
defined surfaces constituted by substantially inorganic materials
including metals, semiconductors, and/or insulators, to which biological
materials selectively adhere in differential manners depending on the
natures of the particular surfaces and biological materials. Following
adhesion of biological materials to the apparatus, such materials can be
optically and electronically and otherwise analyzed in order to detect,
identify and characterize the materials. By "substantially inorganic"
herein is meant that the predominant components of the surface
compositions do not comprise organic materials. However, it is to be
understood that the incorporation of minor concentrations of organic
materials that do not materially detract from the selective bonding
affinity of the substantially inorganic materials employed, is within the
scope of these teachings. By "organic" is meant a composition comprising
a carbon chain.

[0033] In one embodiment, optical characteristics of each of the bottom
surfaces 138, 140, 142, 144, 146, 148, 150, 152 and 154 are recorded as
control data in the absence of a test solution. For example, the optical
characteristics can be determined using equipment suitable for detecting
and recording the optical absorption and reflectance of each of the
bottom surfaces 138, 140, 142, 144, 146, 148, 150, 152 and 154. In this
regard, the apparatus 100 desirably includes test cells 102, 104, 106,
108, 110, 112, 114, 116 and 118 arranged in a regular array. The test
cells are carefully aligned for reading by corresponding equipment
suitable for detecting and recording the optical absorption and
reflectance of each of the bottom surfaces 138, 140, 142, 144, 146, 148,
150, 152 and 154. It is understood that the vertical alignment of the
test cells 102, 104, 106, 108, 110, 112, 114, 116 and 118 in a column is
merely exemplary. For example, analogous test cell arrays can comprise
horizontal rows as well as multiple rows and columns, or other regular
arrays such as test cells arranged in concentric circles. Test cells can
also be individually configured and analyzed.

[0034] A test solution comprising an unknown amino acid or polypeptide is
applied to the respective bottom surfaces 138, 140, 142, 144, 146, 148,
150, 152 and 154 of the test cells 102, 104, 106, 108, 110, 112, 114, 116
and 118. After allowing the passage of a suitable time period for any
bonding of the test solution components on the bottom surfaces to occur,
such as about three (3) hours, the test solution is removed from the test
cells 102-118 and the test cells are rinsed several times using a test
solution solvent. If present in the test solution, an unknown amino acid
or polypeptide will selectively bond to some or all of the bottom
surfaces 138, 140, 142, 144, 146, 148, 150, 152 and 154. The optical
characteristics of the apparatus 100 are then determined, using the same
equipment for detecting and recording the optical absorption and
reflectance of each of the bottom surfaces. Changes in such absorption
and reflectance on some or all of the bottom surfaces 138, 140, 142, 144,
146, 148, 150, 152 and 154 are then computed by comparison with the
corresponding control data. Such changes in optical absorption and
reflectance on the bottom surfaces 138, 140, 142, 144, 146, 148, 150, 152
and 154 collectively constitute a signature for identification of a
particular amino acid or polypeptide present in the test solution. For
example, known samples of monomers or polypeptides of each of the twenty
amino acids lysine (Lys), arginine (Arg), histidine (His), aspartic acid
(Asp), glutamic acid (Glu), threonine (Thr), serine (Ser), asparagine
(Asn), glutamine (Gln), tyrosine (Tyr), proline (Pro), methionine (Met),
cysteine (Cys), tryptophan (Trp), glycine (Gly), alanine (Ala), valine
(Val), isoleucine (Ile), leucine (Leu), and phenylalanine (Phe) can
separately be subjected to these same steps. Such known polypeptides are
commercially available, for example, from Anaspec Inc., San Jose, Calif.
These polypeptides can be made by solid state synthesis. Background
information is provided in Merryfield, R. B., J. Am. Chem. Soc., Vol. 85,
pp. 2149+(1963), the entirety of which hereby is incorporated herein by
reference. The resulting data can be recorded as unique signatures for
each such amino acid or polypeptide. A test solution comprising a given
unknown amino acid or polypeptide can then be identified by comparing the
control signature data to test data computed on the unknowns using the
apparatus 100. In another embodiment, all of the amino acid or
polypeptide solutions can be tagged, such as by fluorescence,
radioactivity, or ligands having known bonding activity. In the latter
case, for example, bonding pairs such as biotin-avidin or
antigen-antibody can be employed. The cells are then developed, such as
by the measurement of fluorescence, radioactivity, or bonding affinity
with marked bonding pair counterparts, and the relative and absolute
strength of bonding in each test cell is read.

[0035] In one embodiment, the following test solution application
procedure was used. An apparatus 100 having a bottom surface with
dimensions of about 2 millimeters by 2 millimeters patterned on a GaAs
substrate was placed in the respective test solution and left for about 3
hours. The apparatus 100 was then removed from the test solution and
rinsed in deionized water for 10 seconds and then dried in nitrogen gas.

[0036] There are four different classes of amino acids as determined by
their side chains, including polar-acidic, polar-basic, polar-neutral,
and non-polar neutral amino acids. The polar-acidic amino acids include
Asp and Glu. The polar-basic amino acids include Lys, Arg and His. The
polar-neutral amino acids include Thr, Ser, Asn, Gln, Tyr and Pro. The
non-polar neutral amino acids include Met, Cys, Trp, Gly, Ala, Val, Ile,
Leu and Phe. Desirably, each of these four groups of amino acids is
considered to have bonding behavior on the bottom surfaces 138, 140, 142,
144, 146, 148, 150, 152 and 154 that is somewhat consistent within the
group. This consistency can aid in identification of test solutions
containing unknown amino acids and polypeptides. In general, the polar
amino acids, including the polar-acidic, polar-basic, and polar-neutral
amino acids, are hydrophilic and accordingly may be soluble in polar
solvents which are used in the test solutions. For example, water can be
used as the solvent. In general, the non-polar neutral amino acids are
hydrophobic and accordingly may be soluble in nonpolar solvents such as
nonpolar hydrocarbons. Polar amino acids may also be somewhat soluble in
nonpolar solvents, and nonpolar amino acids may also be somewhat soluble
in polar solvents.

[0037] The amino acid detection and identification apparatus 100 is
particularly suitable for the testing and identification of individual
amino acids and polypeptides of individual amino acids. In general,
solutions containing more than one amino acid are desirably separated
using conventional techniques before amino acid identification using the
amino acid detection and identification apparatus 100. Separation can be
carried out, for example, using a chromatography column or
electrophoresis gel.

[0038] FIG. 2 shows an array 200 of exemplary control test data for
polypeptides of each of the 20 amino acids, generated using the amino
acid detection and identification apparatus 100. The polypeptides were
separately prepared for each amino acid, generally having chain lengths
of ten (10) amino acid moieties except for minor concentrations of
peptides having chain lengths of eight (8) amino acid moieties. Other
species may be present at insubstantial concentrations. Each polypeptide
further included a 5-carboxyfluorescein (5F-AM) moiety bound at the
exposed --N--H2 group at the polypeptide end, leaving an exposed
--C--O--O--H (carboxylic acid) group at the other end. Although the
exposed --C--O--O--H groups are themselves reactive, this reactivity is
overshadowed by the comparatively greater cumulative reactivity of the
various side chains also present in each amino acid moiety, of which
there accordingly are generally eight (8) or ten (10) in each
polypeptide. Therefore, bonding of the polypeptides to the substantially
inorganic bottom surfaces occurs through these side chains, a
longitudinal side of the polypeptide thus being secured to the bottom
surface. Although some end-bonding of polypeptides through the exposed
--C--O--O--H groups may transiently occur, such bonding is disfavored due
to entropy and other factors, and unlikely to persist. Since each
polypeptide comprises such an exposed --C--O--O--H group, strong bonding
there would lead to indistinguishable results among testing of various
polypeptides. Hence, operation of the amino acid detection and
identification apparatus 100 takes advantage of the dynamics of this
bonding environment to provide test results facilitating differentiation
between polypeptides of different amino acids. In alternative
embodiments, fluorescein, or fluorescein 5-isothiocyanate (FITC), are
used as markers instead of 5F-AM. In the case of Cys polypeptides, the
following peptide sequence was used in view of the potential for
excessive disulfide crosslinking:
5F-AM-Ala-Cys-Ala-Ala-Ala-Cys-Ala-Ala-Ala-OH. A potential source of
variability in the results is the presence of contaminants in the
polypeptides that could induce or block adhesion to the substantially
inorganic surfaces.

[0039] In this exemplary embodiment, each of the 20 polypeptides was
separately dissolved in water to generate the control solutions for
testing. In one embodiment, a 1.0 millimolar concentration of the
polypeptides was used. The left-most column of FIG. 2 shows row headings
for the control test data array. The row headings identify and correspond
to the metals, semiconductors and insulators on the bottom surfaces 138,
140, 142, 144, 146, 148, 150, 152 and 154 respectively of the amino acid
detection and identification apparatus 100. The top-most row of FIG. 2
shows column headings for the control test data array. The column
headings identify and correspond to the individual known polypeptide
solutions that were separately tested as reported in each column of the
control test data array.

[0040] The control test data array shown in FIG. 2 is indicative of the
relative and numerical concentrations of polypeptides bound to the
indicated substantially inorganic surfaces when the test cells of the
amino acid detection and identification apparatus 100 were subjected to
known aqueous solutions of each individual polypeptide. The units of the
numerical data are in 1×103 polypeptides per square micrometer
(μm2). The margin of error in the data was about twenty percent
(20%). This margin of error included both statistical error and
systematic error. Systematic errors include, for example, variations in
results due to differences in the processes for preparation of and of the
concentrations in the polypeptide solutions. The impact of margin of
error effects on the reliability and repeatability of test results can be
moderated by carrying out multiple trials and then averaging the
numerical results.

[0041] The control test cell data for the polar-acidic and polar-basic
polypeptides of Lys, Arg, His, Asp and Glu are grouped together in the
left section of the test cell data array shown in FIG. 2. The test cell
data for the polar-neutral polypeptides of Thr, Ser, Asn, Gln, Tyr and
Pro are grouped together in the middle section of the test cell data
array. The test cell data for the remaining non-polar neutral
polypeptides of Met, Cys, Tip, Gly, Ala, Val, Ile, Leu and Phe are
grouped together in the right section of the test cell data array. Each
of the data in the test cell data array visually and numerically
indicates the degree to which the designated polypeptide in each test
bonded to the designated substantially inorganic surface. For example,
data point 202 shows that an aqueous solution of polar-basic Arg
polypeptide strongly bonded to Al, as indicated both by the dark shading
and the high numerical reading, 61×103/μm2. Data point
203 shows, in contrast, that the Lys polypeptide only lightly bonded to
Al, as indicated both by the light shading and the light reading,
3.5×103/μm2. Further for example, data point 204 shows
that an aqueous solution of polar-acidic Asp polypeptide firmly bonded to
AlGaAs, as indicated both by the medium dark shading and the elevated
numerical reading, 16×103/μm2. Data point 205 shows,
in contrast, that the His polypeptide only lightly bonded to AlGaAs, as
indicated both by the light shading and the light reading,
1.8×103/μm2. Additionally for example, data point 206
shows that an aqueous solution of polar-neutral Thr polypeptide
moderately bonded to SiO2, as indicated both by the grey shading and
the moderate numerical reading, 5.2×103/μm2. Data
point 207 shows, in contrast, that Asn polypeptide only minimally bonded
to SiO2, as indicated both by the lack of shading and the low
numerical reading, 0.9×103/μm2. Furthermore for
example, data point 208 shows that an aqueous solution of non-polar
neutral Gly polypeptide lightly bonded to Si3N4, as indicated
both by the light shading and the light reading,
3.4×103/μm2. Data point 209 shows, in contrast, that
Ala polypeptide only minimally bonded to Si3N4, as indicated
both by the lack of shading and the low numerical reading, less than
(<)0.5×103/μm2. In addition, for example, data
point 210 shows that an aqueous solution of non-polar neutral Met
polypeptide minimally bonded to Pt, as indicated both by the lack of
shading and the low numerical reading, 0.7×103/μm2.

[0042] The visual and numerical test data reflected in the control test
data array 200 can be used to identify the amino acid content of unknown
aqueous polypeptide solutions. The control test data array in FIG. 2
shows the strength of the bonding that results from exposure of each of
the nine substantially inorganic surfaces separately to each of the
twenty amino acid oligomers (polypeptides). The strength of such bonding,
ranging from strong, to firm, moderate, light, and minimal, constitutes
an indication of the amino acid identity as correlated with the data in
FIG. 2. FIG. 2 shows that most of the strongest bonding reactions
occurred with polar-acidic and polar-basic polypeptides, and that the
strongest bonding reactions involved the Si3N4, SiO2,
AlGaAs, and Al surfaces. However, each of the control tests reported in
the array did generate a numerical bonding reading. In addition, each of
the exemplary control tests reported in columns 212, 214 and 216
generated a different series of readings for the nine substantially
inorganic test surfaces. For example, Gly polypeptide in column 214
lightly bonded to Si3N4, SiO2 and AlGaAs, but Ile
polypeptide in column 216 lightly bonded only to SiO2 and AlGaAs,
and instead minimally bonded to Si3N4. These different series
of numerical polypeptide bonding values can be used as signatures to
distinguish Gly from Ile. Further analogous series of numerical bonding
values can be used to identify other polypeptides in a test solution that
is applied to the apparatus 100. The numerical bonding values for a given
polypeptide are generally independent of the concentration of the
polypeptides in solution, provided that bonding surface saturation by
polypeptides occurs. Where, as reported in FIG. 2, oligomers of
individual amino acids are tested, the test data reflect the
concentration of the polypeptides rather than of the individual amino
acid molecules. The units are calibrated to 1×103 amino acid
oligomers per square micrometer.

[0043] FIG. 3 shows another array 300 of exemplary control test data for
oligomers of the 20 amino acids prepared in the same manner as described
above in connection with FIG. 2, generated using the amino acid detection
and identification apparatus 100. In this exemplary embodiment, each of
the twenty amino acid oligomers so tested was constituted in a 0.25 molar
polypeptide solution using 1 Molar
(N-2-[hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (HEPES) diluted
in water, in order to generate the control solutions for testing. As in
FIG. 2, the left-most column of FIG. 3 shows row headings for the control
test data array. The row headings identify and correspond to the metals,
semiconductors and insulators on the bottom surfaces 138, 140, 142, 144,
146, 148, 150, 152 and 154 respectively of the amino acid detection and
identification apparatus 100. The top-most row of FIG. 3 shows column
headings for the control test data array. The column headings identify
and correspond to the individual known polypeptide solutions that were
separately tested as reported in each column of the control test data
array. The numerical data are again reported in units of 1×103
amino acid oligomer molecules per square micrometer. The control test
data array shown in FIG. 3 is indicative of the relative concentrations
of polypeptides bound to the indicated substantially inorganic surfaces
when the test cells of the amino acid detection and identification
apparatus 100 were subjected to known HEPES solutions of each individual
polypeptide.

[0044] Each of the data in the test cell data array 300 visually and
numerically indicates the degree to which the designated polypeptide in
each test bonded to the designated substantially inorganic surface. For
example, data point 302 shows that a HEPES solution of polar-basic Lys
polypeptide strongly bonded to Si3N4, as indicated both by the
dark shading and the high numerical reading,
21×103/μm2. Data point 303 shows, in contrast, that
Ser polypeptide only lightly bonded to Si3N4, as indicated both
by the light shading and the light reading,
2.1×103/μm2. Further for example, data point 304 shows
that a HEPES solution of polar-neutral Thr polypeptide firmly bonded to
SiO2, as indicated both by the medium dark shading and the elevated
numerical reading, 12×103/μm2. Data point 305 shows,
in contrast, that Asn polypeptide only lightly bonded to SiO2, as
indicated both by the light shading and the light reading,
2.3×103/μm2. Additionally for example, data point 306
shows that a HEPES solution of non-polar neutral Met polypeptide
moderately bonded to Si3N4, as indicated both by the grey
shading and the moderate numerical reading,
4.4×103/μm2. Data point 307 shows, in contrast, that
Gln polypeptide only lightly bonded to Si3N4, as indicated both
by the light shading and the light reading,
1.8×103/μm2. Furthermore for example, data point 308
shows that a HEPES solution of non-polar neutral Gly polypeptide lightly
bonded to SiO2, as indicated both by the light shading and the light
reading, 3.0×103/μm2. Data point 309 shows, in
contrast, that Ala polypeptide only minimally bonded to SiO2, as
indicated both by the lack of shading and the low numerical reading,
<0.5×103/μm2. In addition, for example, data point
310 shows that a HEPES solution of non-polar neutral Ala polypeptide
minimally bonded to Al, as indicated both by the lack of shading and the
low numerical reading, 0.7×103/μm2. FIG. 3 shows the
strength of the bonding that resulted from exposure of each of the nine
substantially inorganic surfaces separately to each of the twenty amino
acid oligomers. As in the case of FIG. 2, FIG. 3 shows that most of the
strongest bonding reactions occurred with polar-acidic and polar-basic
polypeptides, and that the strongest bonding reactions involved the
Si3N4, SiO2, AlGaAs, and Al surfaces. However, each of the
test cells did generate a numerical data reading. In addition, each of
the exemplary test data columns 312, 314, and 316 generated a different
series of readings for the nine test surfaces. For example, Ile
polypeptide in column 314 moderately bonded to Al and lightly bonded to
SiO2, but Leu polypeptide in column 316 only lightly bonded to Al,
and minimally bonded to SiO2. These differential bonding patterns
can be used to distinguish Ile from Leu. Further differential bonding
patterns potentially can be mapped from the FIG. 3 data and used to
distinguish any two HEPES amino acid oligomer solutions from each other
in a likewise manner.

[0045]FIG. 4 shows a further array 400 of exemplary control test data for
oligomers of the 20 amino acids prepared in the same manner as described
above in connection with FIG. 2, generated using the amino acid detection
and identification apparatus 100. In this exemplary embodiment, each of
the twenty amino acid oligomers so tested was constituted in a 0.25 molar
polypeptide solution using undiluted dimethyl sulfoxide (DMSO) in order
to generate the control solutions for testing. As in FIGS. 2 and 3, the
left-most column of FIG. 4 shows row headings for the control test data
array. The row headings identify and correspond to the metals,
semiconductors and insulators on the bottom surfaces 138, 140, 142, 144,
146, 148, 150, 152 and 154 respectively of the amino acid detection and
identification apparatus 100. The top-most row of FIG. 4 shows column
headings for the control test data array. The column headings identify
and correspond to the individual known amino acid oligomer solutions that
were separately tested as reported in each column of the control test
data array. The numerical data were again reported in units of
1×103 amino acid oligomer molecules per square micrometer. The
control test data array shown in FIG. 4 is indicative of the relative
concentrations of polypeptides bound to the indicated substantially
inorganic surfaces when the test cells of the amino acid detection and
identification apparatus 100 were subjected to known DMSO solutions of
each individual polypeptide. In another embodiment, polypeptides were
solubilized in a 1:5 DMSO:water solution at a 1 millimolar polypeptide
concentration. Higher concentrations of DMSO can be beneficial in
solubilizing polypeptides of Tyr, Phe and Leu.

[0046] Each of the data in the test cell data array 400 visually and
numerically indicates the degree to which the designated polypeptide in
each test bonded to the designated substantially inorganic surface. For
example, data point 402 shows that a DMSO solution of polar-basic Arg
polypeptide strongly bonded to Si3N4, as indicated both by the
dark shading and the high numerical reading,
21×103/μm2. Data point 403 shows, in contrast, that
Thr polypeptide only lightly bonded to Si3N4, as indicated both
by the light shading and the light reading,
2.1×103/μm2. Further for example, data point 404 shows
that a DMSO solution of polar-acidic Glu polypeptide firmly bonded to
AlGaAs, as indicated both by the medium dark shading and the elevated
numerical reading, 10×103/μm2. Data point 405 shows,
in contrast, that Asp polypeptide only lightly bonded to AlGaAs, as
indicated both by the light shading and the light reading,
3.7×103/μm2. Additionally for example, data point 406
shows that a DMSO solution of polar-neutral Thr polypeptide moderately
bonded to Al, as indicated both by the grey shading and the moderate
numerical reading, 4.4×103/μm2. Data point 407 shows,
in contrast, that Asn polypeptide only minimally bonded to Al, as
indicated both by the lack of shading and the low numerical reading,
0.7×103/μm2. Furthermore for example, data point 408
shows that a DMSO solution of non-polar neutral Cys polypeptide lightly
bonded to SiO2, as indicated both by the light shading and the light
reading, 3.4×103/μm2. Data point 409 shows, in
contrast, that Met polypeptide moderately bonded to SiO2, as
indicated both by the moderate shading and the moderate reading,
4.6×103/μm2. In addition, for example, data point 410
shows that a DMSO solution of non-polar neutral Met polypeptide minimally
bonded to Au, as indicated both by the lack of shading and the low
numerical reading, 0.9×103/μm2.

[0047]FIG. 4 shows the strength of the bonding that results from exposure
of each of the nine substantially inorganic surfaces separately to each
of the twenty amino acid oligomers. As in the case of FIGS. 2 and 3, FIG.
4 shows that most of the strongest bonding reactions occurred with
polar-acidic and polar-basic amino acid oligomers, and that the strongest
bonding reactions involved the Si3N4, SiO2, AlGaAs, and Al
surfaces. However, each of the test cells did generate a numerical
reading. In addition, each of the exemplary test data columns 412, 414,
and 416 generated a different series of readings for the nine test
surfaces. For example, Ala polypeptide in column 414 lightly bonded to
SiO2 and only minimally bonded to Si3N4, but Phe
polypeptide in column 416 lightly bonded to both SiO2 and
Si3N4. These differential bonding patterns can be used to
distinguish Ala from Phe. Further differential bonding patterns can be
mapped from the FIG. 4 data and used to distinguish any two aqueous amino
acid oligomers from each other in a likewise manner.

[0048] The preceding discussion in connection with FIGS. 1-4 has been
directed to substantially inorganic surfaces made from the metals Pd, Au,
Ti, Pt, and Al; the semiconductors GaAs, and AlGaAs; and the insulators
Si3N4, SiO2. It is to be understood, however, that other
metals, semiconductors and insulators can be used in addition to or in
substitution for one or more of the substantially inorganic surfaces
addressed in FIGS. 1-4. In addition, alloys or mixtures of two or more
such metals, semiconductors and insulators, and mixtures of one or more
metals, semiconductors, and/or insulators can be used. Each of such
materials will have its own characteristic pattern of bonding affinity
for individual amino acids and polypeptides. These bonding affinities can
be mapped in the same manner as discussed above in connection with FIGS.
1-4, and amino acid detection and identification apparatus can be
constructed and used in the same manner.

[0051] In general, any substantially inorganic insulator can be used,
alone or together with other insulators, metals, and/or semiconductors,
in a surface for selective amino acid or polypeptide bonding. In one
embodiment, further substantially inorganic insulators that can be so
used, in addition to Si3N4, and SiO2, include: aluminum
oxide (Al2O3), zinc oxide (ZnO), beryllium oxide (BeO), ferrite
(Fe3O4), zirconium oxide (ZrO2), boron carbide (B4C),
silicon carbide (SiC), magnesium diboride (MgB2), and in general,
metallic oxides, carbides, borides, nitrides, and sulfides.

[0052] FIGS. 2, 3 and 4 respectively employed water, HEPES diluted in
water, and DMSO as a solvent for the polypeptides, forming solutions of
such amino acid oligomers. Although the term "solution" is used
throughout this discussion, it is to be understood that amino acid
oligomers can alternatively be mobilized in other forms in fluids, such
as, for example, dispersions, suspensions, gels, emulsions, and aerosols.
Furthermore, water, HEPES diluted in water, and DMSO are exemplary
solvents and fluid vehicles, and other solvents and fluid vehicles as
suitable for the fluid mobilization of the polypeptides, proteins, or
other amino acid-comprising compositions can also be used. Polar solvents
such as water preferably dissolve polar-acidic, polar-basic and
polar-neutral amino acids and polypeptides. Non-polar organic solvents
preferentially dissolve non-polar neutral amino acids and polypeptides.

[0053]FIG. 5 shows summary bar graph data based on the tests carried out
to generate FIGS. 2-4, plotting the density on GaAs, Si3N4,
SiO2, Al and Pd surfaces, of equivalent bound
polypeptides×103 per square micrometer (μm2), as to
each of the twenty amino acid oligomers. FIG. 5 shows that the strongest
bonding interactions occurred when Si3N4 and SiO2 surfaces
were exposed to solutions of polar-basic polypeptides. FIG. 5 further
shows that polar-acidic polypeptides generally adhered strongly to
Si3N4 and SiO2, although not as strongly as did the
polar-basic polypeptides. FIG. 5 also shows that polar-basic and
polar-acidic polypeptides generally adhered firmly to Al, although not as
strongly as to Si3N4 and SiO2. FIG. 5 additionally shows
that polar-neutral and non-polar neutral polypeptides generally adhered
moderately to Si3N4, SiO2, and Al, although not as
strongly as did polar-basic and polar-acidic polypeptides. FIG. 5
furthermore shows that all types of polypeptides, including polar-basic,
polar-acidic, polar-neutral, and non-polar neutral polypeptides,
generally adhered at least minimally or lightly to GaAs and Pd. FIG. 5
makes clear that the relative bonding affinity of polypeptides to the
five exemplary substantially inorganic surfaces can be used in either a
quantitative or relative qualitative manner together with known controls
in order to identify particular polypeptides in solution.

[0054] FIG. 6 shows graphed data plotting on the y-axis the adhered
density, on Si3N4 surfaces, of equivalent bound
polypeptides×103 per μm2 including Glu, His, and Lys,
and on the x-axis a pH range of between about 5.5 and about 11.75. The pH
can be increased, for example, by addition of NH4OH. As to Glu, the
density of equivalent bound polypeptides remained stable at about 27,000
per μm2 of surface across a pH range of between about 5.5 and
about 6.0; and gradually dropped to a minimal density that was then
maintained at a pH above about 6.2. As to His, the density of equivalent
bound polypeptides remained stable at about 22,000 per μm2 of
surface across a pH range of between about 5.2 and about 7.2; and
gradually dropped to a minimal density that was then maintained at a pH
above about 7.3. As to Lys, the density of equivalent bound polypeptides
remained stable at about 28,000 per μm2 of surface across a pH
range of between about 6.2 and about 10.0; and gradually dropped to a
minimal density then maintained at a pH above about 10.6. Similar effects
can be demonstrated for the other polar-charged polypeptides, including
polar-basic Arg and polar-acidic Asp. Ionization behavior of the
polar-neutral polypeptides can also be used to affect the bonding
affinity of these amino acid oligomers to substantially inorganic
surfaces.

[0055] FIG. 7 shows graphed data plotting on the y-axis the adhered
density on Si3N4 surfaces, of equivalent bound His
polypeptides×103 per μm2, and on the x-axis a
polypeptide concentration range of between about 1×10-4
millimolar (thousandths of a mole of polypeptides per liter) (mM/l) and
about 1×10-1 mM/l. All concentrations are expressed as
equivalent polypeptides in millimoles. FIG. 7 shows that for polypeptide
solutions having amino acid oligomer concentrations of less than about
5×10-3 mM/l, no appreciable bonding to the substantially
inorganic surfaces occurs. Over a concentration range of between about
5×10-3 mM/l and about 1×10-2 mM/l, the density of
adhered polypeptides steadily increases to about 24,000 equivalent bound
polypeptides per μm2, and this density is then maintained at
higher polypeptide concentrations. Hence, FIG. 7 shows that a given
substantially inorganic surface has a limited capacity for bonding
polypeptides before saturation occurs.

[0056] FIG. 8 shows a schematic side view of an embodiment of an amino
acid detection and identification apparatus 800 designed to selectively
bond a particular polypeptide, and shows such a polypeptide 802 suspended
over the apparatus 800. In this embodiment, the apparatus 800 is designed
to selectively bond a polypeptide comprising five (5) Asp molecules
forming a polypeptide chain indicated at 806, the polypeptide having
either three (3) or five (5) Leu molecules further extending the
polypeptide chain at both ends. The amino acid detection and
identification apparatus 800 comprises a midlayer of AlGaAs 805
interposed between two layers of GaAs 810 and 815. The GaAs layers 810
and 815 are visually distinguished by cross-hatching. The thickness of
the AlGaAs midlayer 805 is about 1.9 nanometers (nm) as indicated by the
double arrow 820. This thickness is less than or equal to the
longitudinal length of the polypeptide consisting of five Asp molecules
indicated at 806.

[0057] In one embodiment, the thickness of the GaAs layer 810 is about 1.7
nm as indicated by the double arrow 825. This thickness approximately
matches or exceeds the longitudinal length of a polypeptide 826
consisting of three Leu molecules having end-bonded 5-carboxyfluorescein
(5F-AM). Fluorescein is a fluorescent marker that enables detection of a
bound polypeptide on the amino acid detection and identification
apparatus 800. The 5F-AM marker extends the longitudinal length of the
polypeptide itself. The thickness of the GaAs layer 815 is about 1.2 nm
as indicated by the double arrow 830. This thickness approximately
matches or slightly exceeds the longitudinal length of a polypeptide 827
consisting of three Leu molecules.

[0058] In an alternative embodiment, the thickness of the GaAs layer 810
is about 2.5 nm as indicated by the double arrow 825. This thickness
approximately matches or exceeds the longitudinal length of a polypeptide
consisting of five Leu molecules 826 having end-bonded 5F-AM. The
thickness of the GaAs layer 815 is about 2 nm as indicated by the double
arrow 830. This thickness approximately matches or exceeds the
longitudinal length of a polypeptide 827 consisting of five Leu
molecules.

[0059] In a further alternative embodiment, the AlGaAs midlayer 805 is
extended to include region 807 indicated by a dotted line in FIG. 8. In
this embodiment, the double arrow 821 indicates the distance j between
the AlGaAs midlayer 805 and the polypeptide 806 comprising five (5) Asp
molecules. Additionally in this embodiment, the double arrow 822
indicates the distance k between the GaAs layer 810 and the polypeptide
consisting of five Leu molecules 826 having end-bonded 5F-AM. Further in
this embodiment, the double arrow 823 indicates the distance 1 between
the GaAs layer 815 and the polypeptide consisting of five Leu molecules
827 having an exposed --C--O--O--H end group. It can be seen that the
distance j is less than the distances k and l. Stated otherwise, the
region 807 constitutes a shelf on which the polypeptide 806 can rest.
This shelf, and the greater distances k and l, provide extra space on
which the polypeptides 826 and 827 can rest when the polypeptide 806
becomes bonded to region 807 of the AlGaAs midlayer 805. In this manner,
the polypeptide 806 can more readily bond to the AlGaAs midlayer 805
without steric hindrance between the side groups of the Leu moieties and
the GaAs layers 810 and 815.

[0060] In use, the amino acid detection and identification apparatus 800
is exposed to a solution of polypeptides. If any of the polypeptides
include chains comprising Leu-Leu-Leu-Asp-Asp-Asp-Asp-Asp-Leu-Leu-Leu, or
a similar polypeptide with two additional Leu molecules at each end of
the chain, then such polypeptides will selectively bond to the amino acid
detection and identification apparatus 800 having layers 805, 810 and 815
of the appropriate thickness discussed above. This bonding will occur in
alignment with the arrows 816, 817, 818 and 819. Polypeptides having
additional chain portions beyond the selectively bonded polypeptide will
adhere only such selected polypeptide, leaving the other chain portions
unbonded and trailing away from the apparatus 800. Selective polypeptide
bonding can be detected, for example, by labeling the polypeptides with
5F-AM as discussed earlier. It will be understood that a given apparatus
800 may be capable of bonding more than one specific polypeptide
sequence, as the various bonding surface materials often have bonding
affinities for more than one amino acid.

[0061] The term "layer" as used throughout this specification is defined
as a body of the subject material as applied over an adjoining surface,
however such body is formed. A "layer" may have a non-uniform thickness,
does not have to be completely continuous, and may be the result of any
desired deposition process undertaken in one or more than one steps.
Hence, a "layer" may also comprise multiple layers of the same or
different materials, which may or may not interpenetrate each other, and
which layers together are referred to as the "layer". There is no
particular limitation on the thickness of a layer except as stated.

[0062] FIG. 9 schematically illustrates the importance of controlling the
thicknesses of the layers in the amino acid detection and identification
apparatus 800, in order to maximize the bonding potential for the
polypeptide 802. The four images 900 represents schematic side views of a
progressive series of amino acid detection and identification apparatus
905, 910, 915 and 920 similar to the amino acid detection and
identification apparatus 800. Regions in the apparatus 905, 910, 915 and
920 distinguished by cross-hatching indicate GaAs layers 902, which are
interposed by AlGaAs layers 904 without cross-hatching, similar to the
structure of the apparatus 800 shown in FIG. 8. Each of the apparatus
905, 910, 915 and 920 has more than one bonding site for
Leu-Leu-Leu-Asp-Asp-Asp-Asp-Asp-Leu-Leu-Leu, or for a similar polypeptide
with two additional Leu molecules at each end of the chain. In the
apparatus 905, the GaAs layers between any two adjacent polypeptide
bonding sites have a thickness d, indicated by the double arrow 925. The
thickness d is more than adequate to prevent steric interference between
adjacently bonded polypeptides. In the apparatus 910, the GaAs layers
between any two adjacent polypeptide bonding sites have a thickness e
indicated by the double arrow 930 that is still adequate to prevent
steric interference between adjacently bonded polypeptides, but some of
the polypeptides nearly butt ends as at exemplary point 935. In the
apparatus 915, the GaAs layers between any two adjacent polypeptide
bonding sites have a thickness f indicated by the double arrow 940 that
is too small to prevent steric interference between adjacent polypeptides
at some of the potential bonding sites. Although some bonding can still
occur, exemplary bonding sites 945 and 950 are sterically blocked,
reducing the selective bonding capacity of the apparatus 915. In the
apparatus 920, the distance between the AlGaAs layers of any two adjacent
polypeptide bonding sites constitutes a thickness g indicated by the
double arrow 955 that is too small to allow any selective bonding of
polypeptides. At every intended bonding site, steric interference between
adjacent AlGaAs midlayers prevents bonding of any polypeptides at any of
the intended bonding sites. FIG. 9 illustrates the importance of
designing and controlling the thicknesses of substantially inorganic
layers for selective bonding of polypeptides in order to avoid decreased
apparatus capacity or total failure due to steric hindrance factors. In a
further alternative embodiment, the AlGaAs midlayers are extended in the
same manner as discussed above in connection with the midlayer 805 and
region 807 shown in FIG. 8.

[0063] FIG. 10 plots data regarding the bonding of polypeptides on the
amino acid detection and identification apparatus 905-920 shown in FIG.
9. The x-axis denotes the separations d, e, f and g, in nm, between
mutually adjacent AlGaAs layers as shown in FIG. 9. The y-axis denotes
the adhered density of equivalent bound polypeptides×103 per
μm2 on the amino acid detection and identification apparatus
905-920. The circular data plot trials in which the polypeptides applied
to the amino acid detection and identification apparatus 905-920 had
three Leu moieties on the polypeptide ends, each having an end-to-end
length of about 1.2 nm. The square data plot trials in which the
polypeptides applied to the amino acid detection and identification
apparatus had five Leu moieties on the polypeptide ends, each having an
end-to-end length of about 2 nm. In each case, the polypeptides were
labeled at one end with 5F-AM. Referring first to the circular data
points, the adhered density of
Leu-Leu-Leu-Asp-Asp-Asp-Asp-Asp-Leu-Leu-Leu polypeptides steadily
increased as the separation between the AlGaAs layers was reduced from
about 3.4 nm to about 2.5 nm to about 1.4 nm, and then dramatically
dropped as the separation was reduced to about 1.2 nm. The steady
increase evident in the first three such data points indicates that there
was no significant steric hindrance to polypeptide binding, while the
packing density of binding sites became correspondingly greater. The
sudden drop in polypeptide adhesion density at an AlGaAs layer separation
of about 1.2 nm indicates that substantial steric hindrance to
polypeptide bonding arose at this smaller layer separation. A similar
pattern resulted in the square data points regarding the adhered density
of Leu-Leu-Leu-Leu-Leu-Asp-Asp-Asp-Asp-Asp-Leu-Leu-Leu-Leu-Leu. In each
case, the polypeptides were labeled at one end with 5F-AM. There, the
polypeptide adhesion density increased as the AlGaAs layer separation was
decreased from about 3.4 nm to about 2.5 nm. However, the adhesion
density at an AlGaAs layer separation of about 1.4 nm was almost as low
as that at a separation of about 1.2 nm. This result is consistent with
the additional chain length of the polypeptides being selectively bonded
in the square plotted data, because the additional polypeptide length
caused steric hindrance to first arise at a greater AlGaAs layer
separation. The results of the trials reported in FIG. 10 further
illustrate the trend in bonding performance through the progression in
amino acid detection and identification apparatus 905, 910, 915 and 920,
as discussed in connection with FIG. 9.

[0064] FIG. 11 shows a schematic perspective view of an embodiment of an
amino acid detection and identification apparatus 1100. The apparatus
1100 comprises three sandwiched substantially inorganic layers 1105, 1110
and 1115, each of which may be independently selected from among the
substantially inorganic metals, semiconductors and/or insulators and
mixtures as earlier discussed. The optimal compositions of the layers
1105, 1110 and 1115 are determined by the polypeptide to be selectively
bonded to and thus made detectable by the apparatus 1100. Referring back
to FIG. 8, the selected polypeptide will then bond across layers 1105,
1110 and 1115 in the same manner as the exemplary
Leu-Leu-Leu-Asp-Asp-Asp-Asp-Asp-Leu-Leu-Leu polypeptide bonded across
layers 805, 810 and 815 discussed in connection with FIG. 8. One
advantage of the structure of the apparatus 1100 is that the layers 1105,
1110 and 1115 can be successively built up on a non-bonding substrate
1120, and then etched to reveal layers with the selected bonding
activity. In one embodiment, the thus exposed layers 1105, 1110 and 1115
are mutually flush so that a selected polypeptide will bond to all three
layers. In another embodiment, one or more of the layers may be recessed
or formed from a non-bonding material that serves as a spacing element
rather than a bonding surface. In one embodiment, only the layer 1110
serves as a bonding surface for amino acids. In one modification of that
embodiment, conductors 1125 and 1130 are in electrical communication with
an external source for applying a voltage potential across the layer
1110. In this manner, a change in conductivity across the layer 1110
detected by the external voltage source is an indication of selective
bonding of amino acids or polypeptides on the layer 1110. Although the
exemplary apparatus 1100 comprises three layers 1105, 1110 and 1115, any
desired number of bonding and/or spacing layers can be built up and
exposed to selectively bond a desired polypeptide sequence. It will be
understood that a given apparatus 1100 may be capable of bonding more
than one specific polypeptide sequence, as the various surface materials
often have bonding affinities for more than one amino acid. Apparatus can
be designed to carry out the same operations with regard to
macromolecules comprising amino acids, such as proteins.

[0065] FIG. 12 shows an embodiment of an amino acid detection and
identification apparatus 1200 which is suitable for the detection and
identification of macromolecules comprising amino acids. The apparatus
1200 comprises AlGaAs surface site layers 1205, 1210, and 1215 interposed
between GaAs interlayers 1220, 1225, 1230 and 1235. Distal site ends
1240, 1245 and 1250 of the AlGaAs surface site layers 1205, 1210, and
1215 extend beyond ends of the GaAs interlayers 1220, 1225, 1230 and
1235, forming a polypeptide bonding region 1227. All of the foregoing
layers are sandwiched between AlGaAs support layers 1255 and 1260.
Conductors 1265 and 1270 are provided on surfaces of the AlGaAs support
layers 1255 and 1260 adjacent to the polypeptide bonding region 1227. The
AlGaAs support layers 1255 and 1260 serve to position the conductors 1265
and 1270 adjacent to the polypeptide bonding region 1227, and to form the
bonding region 1227 as a well for containing a test solution potentially
containing a polypeptide macromolecule. A target polypeptide
macromolecule 1285 having regions that selectively bond with AlGaAs,
desirably located in precise alignment with the distal site ends 1240,
1245 and 1250, will then selectively bond on surfaces of the distal site
ends to the apparatus 1200. The conductors 1265 and 1270 may be in
electrical communication with an external voltage source for applying a
potential across the bonding region 1227 between distal ends 1275 and
1280 of the conductors 1265 and 1270 respectively, for confirming the
presence of a selectively bound polypeptide macromolecule on the
apparatus 1200. A change in the conductivity across the bonding region
1227 is an indication of such presence. Alternatively, for example, the
conductors 1265 and 1270 can be substituted by optical waveguides such as
optical fibers or optical planar waveguides mutually aligned for light
transmission and directing light across the bonding region 1227 so that a
change in transmitted light through such optical waveguides is an
indication of the selective bonding of a polypeptide macromolecule. In
use, a solution potentially comprising the target polypeptide
macromolecule is placed in the vicinity of the region 1227. If present in
the solution, a target polypeptide macromolecule 1285 then selectively
bonds to the apparatus 1200. FIG. 13 shows the same apparatus 1200 in
perspective view. FIG. 13 shows the location of the conductors 1265 and
1270, and exemplary AlGaAs surface site layers 1205 and 1210.

[0066] FIG. 14 shows an apparatus 1400 embodying modifications of the
apparatus 1200. The modifications enable the controlled and independent
application of two different voltages to precisely located regions of a
selectively bound polypeptide macromolecule 1285. In this embodiment,
each of the AlGaAs surface site layers 1205, 1210 and 1215 is formed from
an electrical conductor or semiconductor having a charge carrier
mobility, optionally p-doped or n-doped. The layers 1205 and 1215 are in
electrical contact with conductor 1290, and the layer 1210 is in
electrical contact with conductor 1295. Accordingly, a first voltage V1
can be applied to the layers 1205 and 1215 through conductor 1290, and a
second voltage V2 can independently be applied to the layer 1210 through
conductor 1295. Application of such voltages can be used to modulate the
binding of the target polypeptide macromolecule as a further aid in its
detection and/or identification.

[0067] FIG. 15 shows an application of the apparatus 1200 for the
detection and identification of a target polypeptide macromolecule 1285,
employing an antibody to the target polypeptide macromolecule. In this
embodiment, an antibody 1297 is provided having specific binding affinity
for a target polypeptide macromolecule 1285 constituting an antigen. The
antibody 1297 is anchored within the region 1227 by a polypeptide chain
1298. The polypeptide chain 1298 is selectively bonded to exemplary
layers 1205 and 1210. The polypeptide chain 1298 is bonded to the
antibody 1297 at their interface as indicated by the dotted line 1299.
The polypeptide chain 1298 comprises a polypeptide subregion having a
specific binding affinity for the exemplary layers 1205 and 1210. In use,
the antibody is selectively bonded to the region 1227 by the layers 1205
and 1210. A solution that potentially includes the target polypeptide
macromolecule 1285 is then placed in the vicinity of the region 1227. If
the target polypeptide macromolecule 1285 is present in the solution, a
target macromolecule 1285 selectively bonds to the antibody 1297 secured
to the region 1227 of the apparatus 1200.

[0068] FIG. 16 shows an embodiment of an additional amino acid detection
and identification apparatus 1600 which is suitable for the selective
detection and identification of an amino acid, polypeptide, or
macromolecule comprising amino acids. The apparatus 1600 comprises an
electrically conductive comb 1602 comprising tines 1604, 1606, 1608,
1610, 1612, 1614, 1616 and 1618. The apparatus 1600 further comprises an
electrically conductive comb 1603 comprising tines 1605, 1607, 1609,
1611, 1613, 1615, 1617, 1619, and 1621. The tines 1604-1618 of the comb
1602 are interlaced with and separated by small distances from the tines
1605-1621 of the comb 1603. Pad 1630 is in electrical communication with
the comb 1602; and pad 1632 is in electrical communication with the comb
1603. The pads provide a surface of adequate size for the application of
an externally generated voltage potential, such as by touching
electrically charged probes to the pads. The combs 1602 and 1603 are made
from two independently selected electrically conductive materials
comprising substantially inorganic metals, semiconductors and/or
insulators as earlier discussed. Although the combs are not fabricated
solely from insulators, they can be fabricated from materials comprising
insulators together with metals and/or semiconductors. The combs 1602 and
1603 are separated by substrate 1634. The electrical conductivity of the
substrate 1634 is adequately reduced relative to that of the combs 1602
and 1603 such that a voltage potential across a gap between the combs
1602 and 1603 can be generated. The small distances between the tines of
the combs are then designed and precisely fabricated in a manner
analogous to the manner in which the AlGaAs layer 805 is prepared as
discussed above in connection with FIGS. 8-10. In use, a solution of an
amino acid, polypeptide, or macromolecule comprising amino acids is
applied to the tines 1604-1621 of the combs 1602 and 1603 and to the gap
between them across the substrate 1634. In one embodiment, the minimum
path length across the gap is within a range of between about 200
Angstroms ({acute over (Å)}) and about 2000{acute over (Å)}.
Target amino acids, polypeptides, or macromolecules with a bonding
affinity for the alternating surfaces of the tines in combs 1602 and 1603
will selectively bond to the apparatus 1600. Following removal of the
solution of unbonded amino acids, polypeptides or other macromolecules,
the charge carrier conductivity of the combs 1602 and 1603 can be tested.
A change in conductivity indicates bonding of the amino acids,
polypeptides, or macromolecules.

[0069] FIG. 17 shows an embodiment of a method 1700 for making the amino
acid detection and identification apparatus 100 as discussed above in
connection with FIGS. 1-4. In a series of steps 1705 and 1710, the amino
acid detection and identification apparatus 100 is fabricated. In step
1705, a substrate is provided for a column of test cells 102, 104, 106,
108, 110, 112, 114, 116 and 118. The substrate may be made from any
material suitable for the fabrication of a supportive base for test
surfaces, such as a polymer, metal, or ceramic. A raised outer boundary
wall 120 is provided on the substrate that is capable of containing a
sample of an amino acid or polypeptide solution. Further raised boundary
walls 122, 124, 126, 128, 130, 132, 134 and 136 are provided on the
substrate, defining and mutually separating the test cells 102-118. The
cells 102-118 define exposed and mutually separated portions of the
substrate. In step 1710, bottom surfaces 138, 140, 142, 144, 146, 148,
150, 152 and 154, respectively, are provided on each of the exposed and
mutually separated portions of the substrate. The bottom surfaces 138-154
each independently comprise a selected inorganic metal, semiconductor,
and/or insulator surface that selectively adheres amino acids. The bottom
surfaces 138-154 can each be prepared on the substrate using any suitable
process, such as evaporation, vapor deposition or electrodeposition. In
this embodiment, the metals Pd, Au, Ti, Pt, and Al; the semiconductors
GaAs and AlGaAs; and the insulators Si3N4 and SiO2, are
used. Accordingly, the bottom surfaces 138, 140, 142, 144, 146, 148, 150,
152 and 154 respectively comprise: GaAs, Si3N4, SiO2,
AlGaAs, Al, Pt, Ti, Au, and Pd. In one embodiment, oxides naturally
formed on the metal surfaces such as aluminum oxide are not removed.

[0070] FIG. 18 further shows steps 1805, 1810, 1815, 1820, and 1825 of a
method 1800 for using the apparatus 100 for detection and identification
of a polypeptide 1830 in a fluid. In step 1805, a selected control
polypeptide composition is deposited in each of the test cells 102, 104,
106, 108, 110, 112, 114, 116 and 118 of the amino acid detection and
identification apparatus 100. As earlier discussed, the polypeptide
compositions can be mobilized in any form of fluid, such as, for example,
solutions, dispersions, suspensions, gels, emulsions, and aerosols.
Furthermore, solvents and fluid vehicles other than water, HEPES diluted
in water, and DMSO can be used. In step 1810, first data are recorded as
to selective affinity of each selected test surface for each control
polypeptide composition. A separate apparatus 100 may be used to carry
out each control test, or an apparatus 100 can be chemically treated to
remove any bound polypeptides and reused. In step 1815, an unknown
polypeptide composition is deposited on an amino acid detection and
identification apparatus 100. In step 1820, second data are recorded as
to selective affinity of each selected test surface for the unknown
polypeptide composition. In step 1825, the second data are correlated
with the first data to detect and identify the unknown polypeptide
composition 1830. It is to be understood that the compositions can
comprise more than one polypeptide or may have been previously treated,
e.g., by chromatography or electrophoresis, to isolate a single
polypeptide for identification.

[0071] FIG. 19 shows an embodiment of a method 1900 for making amino acid
detection and identification apparatus 800 as discussed above in
connection with FIGS. 8 and 9. In a series of steps 1905, 1910, 1915, and
1920, the amino acid detection and identification apparatus 800 is
fabricated. In step 1905, a first plurality of surface sites 810 are
provided, each comprising a first substantially inorganic surface
selected from the group consisting of metals, semiconductors, insulators,
and mixtures, each of said first surfaces having selective affinity for
bonding of a portion of a polypeptide. In step 1910, a second plurality
of surface sites 805 are provided, each comprising a second substantially
inorganic surface selected from the group consisting of metals,
semiconductors, insulators, and mixtures, each of said second surfaces
having selective affinity for bonding of a portion of a polypeptide. In
another embodiment, in step 1915, a third plurality of surface sites 815
are provided, each comprising a third substantially inorganic surface,
the second plurality of surface sites 805 interposed between and adjacent
to the first and third pluralities 810 and 815, and having a thickness
for spacing the first and third pluralities of surface sites apart by a
distance suitable for selectively bonding another portion of a
polypeptide to the second plurality of surface sites 805. The first,
second and third pluralities of surface sites 805, 810 and 815 can be
fabricated, for example, as successively deposited layers on a substrate,
not shown, which can for example be interfaced with the side 812 of the
layer 810. In one embodiment, in step 1920 each of the first plurality of
surface sites 810 is provided with a first substantially inorganic
surface extending for a distance away from the adjacent second surface
site 805, the distance being suitable for selectively bonding a portion
of a polypeptide to each of the first plurality of surface sites 810.

[0072] The deposition steps in FIG. 19 can be carried out, for example,
using a vapor deposition process such as molecular beam epitaxy (MBE).
Other vapor deposition techniques, such as plasma enhanced chemical vapor
deposition (PECVD) can also be used. The AlGaAs layers can be selectively
exposed using an etch of H2O2/NH4OH followed by cleaning
with an oxygen plasma. In one embodiment, the AlGaAs surface site layers
had thicknesses of about 0.85 nm and the GaAs interlayers had thicknesses
within a range of between about 1.15 nm and about 5.94 nm. To achieve a
total device thickness of about 10 microns, roughly 3,000 alternating
periods of GaAs and AlGaAs could be used. Extensive pauses between
depositions of the alternating periods are advantageously used. After
fabrication of the device periods, the layering is exposed by cleavage in
air. A wet etch of H2O2/NH4OH (500:1) is then applied,
followed by a water rinse and nitrogen drying. The wet etch is selective
to GaAs versus AlGaAs by a factor of at least about 300:1, thus leaving a
set of veins of AlGaAs surface sites protruding above a background of
GaAs. Exposure to water modifies the adhesive properties of AlGaAs when
compared to washes only in organic solvents. In an alternative
embodiment, AlGaAs is selectively applied to a GaAs substrate using
lithographic masking techniques to form the veins. In another embodiment,
the Al content of AlGaAs is used to control the etching. As the Al
content is reduced, the etching activity on the AlGaAs itself increases,
enabling reduction of the AlGaAs thickness.

[0073] In one embodiment, Si3N4 and SiO2 were deposited as
30 nanometer (nm) thick films using plasma enhanced chemical vapor
deposition (PECVD). Photolithography was used to produce patterns on a
micron-length scale, and dry reactive ion etching (RIE) of the
Si3N4 and SiO2 was accomplished with CF4 and
CH3F respectively to reveal the underlying GaAs. The metals Au, Pd,
Pt, Ti and Al were deposited using electron beam- or thermal-evaporation.
The apparatus 800 were exposed to a four (4) minute oxygen plasma etch as
a cleaning step.

[0074] FIG. 20 further shows steps 2005, 2010 and 2015 of a method 2000
for using the apparatus 800 for detection and identification of a
polypeptide 2020 in a fluid. Referring to FIG. 20, the amino acid
detection and identification apparatus 800 is first calibrated with known
polypeptides in step 2005. Next, data are recorded in step 2010 as to
selective affinity of the apparatus 800 for an unknown polypeptide
composition. In step 2015, the experimental data are correlated with the
calibration data to detect and identify the polypeptide 2020.

[0075] FIG. 21 shows an embodiment of a method 2100 for making amino acid
detection and identification apparatus 1200 as discussed above in
connection with FIGS. 12, 13 and 14. In a series of steps 2105, 2110 and
2115, the amino acid detection and identification apparatus 1200 is
fabricated. In step 2105, a first surface site layer 1205 is provided,
formed from a first substantially inorganic composition selected from the
group consisting of metals, semiconductors, insulators, and mixtures,
said first surface site layer having selective affinity for bonding of a
polypeptide. In step 2110, a plurality of interlayers 1220 and 1225 are
provided, between which the first surface site layer 1205 is interposed.
In step 2115, the first surface site layer 1205 is provided with a distal
site end 1240 extending away from the interlayers 1220 and 1225, the
distal site end 1240 comprising a first surface having selective affinity
for bonding of a polypeptide. In one embodiment, steps 2105, 2110 and
2115 are repeated for the fabrication of a second surface site layer
1210. In another embodiment, the first and second surface site layers
1205 and 1210 and their respective interlayers are interposed between
first and second support layers 1255 and 1260 in step 2120, and first and
second conductors 1265 and 1270 are provided on said first and second
support layers in step 2125 including first and second distal conductor
ends 1275 and 1280 mutually aligned at a gap adjacent to said first and
second surface sites. Referring back to FIG. 14, in an alternative
embodiment each of the AlGaAs surface site layers 1205, 1210 and 1215 is
formed from an electrical conductor or semiconductor, optionally p-doped
or n-doped. The layers 1205 and 1215 are then suitably fabricated by
semiconductor masking, deposition and etching steps so as to be placed in
electrical contact with subsequently-formed conductor 1290. Similarly,
the layer 1210 is suitably fabricated by semiconductor masking,
deposition and etching steps so as to be placed in electrical contact
with conductor 1295. For example, in step 2130 metals suitable for
forming conductors compatible with the n- and p-doped semiconductors used
in making a particular device can be diffused through the various layers
to make selective contact with the layers 1205, 1210 and 1215.

[0076] FIG. 22 further shows steps 2205, 2210, 2215, 2220, 2225 and 2230
for using the apparatus 1200 for detection and identification of a
polypeptide macromolecule 1285 in a fluid. In step 2205 a control
polypeptide macromolecule is deposited at the first surface site 1240. In
step 2210, first data are recorded as to selective affinity of the first
surface site 1240 for the control polypeptide macromolecule. In step
2215, the apparatus 1200 is regenerated by removal of any bound control
polypeptide, or an additional amino acid detection and identification
apparatus is provided for testing of a fluid comprising an unknown
polypeptide macromolecule. In step 2220, an unknown polypeptide
macromolecule is deposited at the first surface site 1240. In step 2225,
second data are recorded as to selective affinity of the first surface
site 1240 for the unknown polypeptide macromolecule. In step 2230, second
data are correlated with first data to detect and identify the
polypeptide macromolecule 2235. In an embodiment where the conductors
1290 and 1295 are provided, an external bias can be provided in
electrical communication with such conductors, capable of applying a
voltage potential across the region 1227.

[0077] In one embodiment, the specificity of an apparatus 1200 for bonding
and thus detecting a specific polypeptide macromolecule is tested by
first producing polypeptides having active regions consistent with the
active regions of the macromolecule considered as bonding sites, which
are fluorescently labeled. The capability of the apparatus 1200 to bond
such polypeptides is then tested. When a suitable spatial arrangement of
bonding sites in the apparatus 1200 for bonding such polypeptides is
found, then polypeptides of increased size emulating the local region of
the target macromolecule near the active bonding sites are generated and
tested for bonding capability. Once acceptable bonding is attained, then
bonding of a known sample solution of the target macromolecule is tested.
Atomic force microscopy is then used to detect the bonding of the target
macromolecule, and fluorescent labeling is discontinued. The specificity
of the apparatus 1200 can then be assessed by testing adhesion of known
false positive producing proteins. The structure of the apparatus 1200 is
then adjusted to sterically hinder bonding of such false positive
producing proteins. In embodiments where conductors 1290 and 1295 are
provided, the bonding capabilities of the apparatus 1200 can be adjusted
by modulating voltage biases applied to such conductors in order to
improve the specific bonding affinity of the apparatus 1200 for a target
polypeptide macromolecule. Certain conductor compositions enable charge
carrier mobility predominantly in either p-doped or n-doped
semiconductors. The specificity of this conductivity can be further
utilized to fabricate apparatus 1200 in which external voltage biases
applied to the conductors 1290 and 1295 can be used to selectively modify
the bonding characteristics of the apparatus.

[0078] FIG. 23 shows an embodiment of a method 2300 for making amino acid
detection and identification apparatus 1600 as discussed above in
connection with FIG. 16. In a series of steps 2305, 2310, 2315 and 2320,
the amino acid detection and identification apparatus 1600 is fabricated.
In step 2305, a substrate 1634 formed from an electrically insulating
composition is provided. In step 2310, a first comb 1602 is formed on the
substrate 1634 comprising first tines 1604, 1606, 1608, 1610, 1612, 1614,
1616 and 1618 formed from a first substantially inorganic composition
selected from the group consisting of metals, semiconductors, insulators,
and mixtures, said first tines having selective affinity for bonding of a
polypeptide. In step 2315, a second comb 1603 comprising second tines
1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619 and 1621 is formed from a
second substantially inorganic composition selected from the group
consisting of metals, semiconductors, insulators, and mixtures, said
second tines having selective affinity for bonding of a polypeptide. In
step 2320, desirably carried out simultaneously with steps 2310 and 2315,
the first and second combs 1602 and 1603 are positioned on the substrate
1634 with their respective tines placed in mutually interwoven
relationships at a spaced apart distance that is suitable for traversal
by a polypeptide bonded to, or by multiple polypeptides located between
and separately bonded to, mutually adjacent first and second tines. The
resulting amino acid detection and identification apparatus 1600 can be
used in a manner similar to that discussed in connection with the other
apparatus above.

[0079] It will be recognized that the present teachings may be adapted to
a variety of contexts consistent with this disclosure and the claims that
follow. The apparatus disclosed herein may be designed for selective
bonding affinity with any amino acid-comprising molecules, ranging from
amino acids to macromolecules such as proteins. The substantially
inorganic materials for fabrication of surfaces having bonding affinity
for such molecules broadly include metals, semiconductors and/or
insulators.

Patent applications by Kirk W. Baldwin, Springfield, NJ US

Patent applications by Loren N. Pfeiffer, Harding Township, NJ US

Patent applications by Robert L. Willett, Warren, NJ US

Patent applications by LUCENT TECHNOLOGIES, INC.

Patent applications in class Amino acid or sequencing procedure

Patent applications in all subclasses Amino acid or sequencing procedure